The rabies virus is a rhabdovirus, a nonsegmented RNA virus with negative sense polarity. The genome codes for five proteins: 3 internal proteins are an RNA-dependent RNA polymerase (L), a nucleoprotein (N) and a phosphorylated protein (NS); a matrix protein (M) located on the inner side of the viral envelope and an external surface glycoprotein (G). (Dietzschold, B. & Ertl, H. Cricial Rev. in Immunology 10:427-439, 1991). The virus is transmitted through broken skin by the bite or scratch of an infected animal. This exposure to rabies virus results in its penetration of peripheral unmyelineated nerve endings, followed by spreading through retrograde axonal transport, replication occurring exclusively in the neurons, and finally arrival in the central nervous system (CNS). Infection of the CNS causes cellular dysfunction and ultimately death. (Rupprecht, C. E., & Dietzschold, B. Lab Invest. 57:603, 1987). Since rabies virus spreads directly from cell to cell, it evades immune recognition. (Clark, H. F. & Prabhakar, B. S., Rabies, In: Olson R. G., et al., eds., Comparative Pathology of Viral Disease, 2:165, Boca Raton, Fla.: CRC Press, 1985). Therefore, in order to effectively prevent disease, immunization should inhibit the ability of the virus to enter the cells.
Rabies is a worldwide public health problem. There is no successful treatment of clinical rabies, the outcome is almost always fatal. The rabies virus is maintained in many animal reservoirs, wildlife as well as domestic. Therefore, in order to eliminate pathogenesis in humans, as well as livestock, it is necessary to eliminate these viral reservoirs. The most efficient vaccination protocol would be the development of oral vaccines that induce a long-lasting protection against subsequent exposure to the rabies virus. It has been shown that certain rabies virus variants, such as SAG-2 and SAD B19, or a vaccinia rabies virus glycoprotein recombinant virus are effective vaccines that can be used for the oral vaccination of certain wildlife, such as foxes and raccoons. (Rupprecht, C. E., et al., Emerg. Infect. Dis., 1:107-114, 1995). However, these vaccines do not induce sufficient protective immunity when administered orally to dogs, and it is the domestic dog that is the principal host and major vector of rabies throughout the world. (Fekadu, M., Canine Rabies, In: Baer, G. M., ed. The Natural History of Rabies, 367-378, Boca Raton, Fla.: CRC Press, 1991; Wang, Y. & Walker, P. J., Virology 195:719-731, 1993).
In developing countries, dogs are responsible for ˜94% of human rabies deaths. For example, in Thailand, which has an estimated population of 7 million dogs, one of every 961 dogs was found to test positive for rabies. Assuming a mean vaccination cost of one U.S. dollar per dog, the minimum spending for dog vaccination in developing countries would be around U.S. $50,000,000. (Meslin, F. X., et al., In: Lyssaviruses, Rupprecht, C. E., et al., eds., Springer-Verlang, Berlin, Heidelberg, New York, 1-26, 1994).
In the Americas, the rabies situation is much more complex than that of developing countries. Reservoirs of rabies exist in many diverse wild animal species, in the United States these resevoirs accounted for nearly 93% of the 8513 reported cases of rabies in 1997. (Rupprecht, C. E., et al., Emerging Infectious Diseases 1(4): 107-114, 1995). The most frequently reported rabid wildlife species are raccoons (50.5%), followed by skunks (24.0%). (Rupprecht, C. E., et al., Emerging Infectious Diseases 1(4): 107-114, 1995). Outbreaks of rabies infections in these terrestrial mammals are found in broad geographic areas across the United States. For example, raccoon rabies affects an area of more than 1 million square kilometers from Florida to Maine.
Oral immunization of stray dogs and wildlife against rabies is the most effective method to control, and eventually eradicate, rabies. (Winkler, W. G. & Bogel., K. Sci. Amer., 266(6):86-92, 1992). In this regard, significant progress has been made in the development of oral rabies vaccines for the control of vulpine rabies. (Aubert, M. F. A., et al., Lyssaviruses, Rupprecht, C. E., et al., eds., Springer-Verlang, Berlin, Heidelberg, New York, 219-243, 1994). However, while oral immunization with conventional modified-live vaccines such as SAD B19, SAG-2, or poxvirus-rabies glycoprotein recombinant vaccines are very effective in foxes (Aubert, M. F. A., et al., Lyssaviruses, Rupprecht, C. E., et al., eds., Springer-Verlang, Berlin, Heidelberg, New York, 219-243, 1994), they do not immunize skunks or induce only low seroconversion by the oral route (Rupprecht, C. E., et al., J. Wildl. Dis. 99-102, 1990). Moreover, very high doses of these vaccines (>108.5 TCID50) are necessary to induce protective immunity via oral immunization of dogs. (WHO Report of the 4th WHO consultation on oral immunization of dogs against rabies, Geneva, RabRes, 93:42, 1993). These findings make oral field vaccinations economically impractical. Therefore, in order to control wildlife rabies and rabies in stray dogs worldwide, more potent and cost effective oral rabies vaccines must be developed. There is a high demand for such vaccines. For example, based on previous experience that a minimal density of 20 vaccine-laden baits per square mile is sufficient for immunization of foxes (Aubert, M. F. A., et al., Lyssaviruses, Rupprecht, C. E., et al., eds., Springer-Verlang, Berlin, Heidelberg, New York, 219-243, 1994), more than 20 million doses of vaccine alone would be required for the control of the raccoon rabies enzootic in the Atlantic regions of the United States.
Vaccines prepared with antigenically conserved lab rabies virus strains may not be effective against those found in the wild, i.e., the street virus. There is a need for versatile vaccines suitable for both domestic animals and wildlife, which either serve as reservoirs for human rabies or are economically important species. Efforts have been made to protect free-ranging animals against virulent street virus challenge by oral consumption of a potent vaccine contained within an attractive bait. Yet concerns regarding residual virulence and ineffectiveness remain. Therefore, there exists a long felt need for a new generation of live rabies vaccines. The present invention describes a new generation of live rabies vaccines that has been developed using reverse genetics technology. (Schnell, M. J., et al., EMBO 13:4195-4203, 1994).
In addition to virus-neutralizing antibodies (VNA), which are believed to be the major immune effectors against rabies, rabies virus antigen-specific (CD4+) T helper cells and cytotoxic T cells (CD8+) (Cox, J. H., et al., Infect. Immun. 16:754-759, 1977), as well as innate mechanisms (Hooper, D. C., et al., J. Virol. 72:3711-3719, 1998), play an important role in the immune defense against rabies. The rabies virus glycoprotein (G) induces the production of VNA, while the cellular responses of CD4+ and CD8+ T cells are predominantly triggered by the internal rabies virus proteins; therefore, live rabies virus represents the best immunogen that will confer optimal protective immunity.
The extent of the immune response to immunization with a live virus vaccine is determined by the antigenic mass administered and produced after administration of the vaccine. Inoculation with a live, yet attenuated, virus will allow for the production of antigen in the absence of pathogenicity. The site of antigen production and presentation are also important factors that determine the potency of the vaccine. In this context, the fixed and street rabies virus variants differ substantially in their ability to replicate in neuronal versus non-neuronal cells (neuronal specificity index). (Morimoto, K., et al., J. Neuro Virol., 6:373-381, 2000). The neuronal specificity index of any particular rabies virus variant is determined by its glycoprotein. The glycprotein is also the major viral protein that determines the host specificity of the strain. In this context, it is the rabies glycoprotein that carries the major determinants responsible for the pathogencity of the virus, as well as the determinants that trigger a protective immune response. One aspect of the present invention uses reverse genetics to combine the determinants that render the rabies virus non-pathogenic with the antigenic determinants that are responsible for the elicitation of an effective anti-rabies immune response.
Tissue culture-adapted laboratory and street rabies virus strains differ greatly in their ability to cause a lethal rabies virus encephalitis. (Morimoto, K., et al., J. Neuro Vrol. 6:373-381, 2000). The pathogenicity of individual rabies virus strains for immunocompetent adult mice appears to correlate inversely with their capacity to induce cell death in vitro and in vivo. For example, CVS-N2c, a highly pathogenic variant derived from the mouse-adapted CVS-24 rabies virus strain, was recently shown to induce significantly less apoptosis in primary hippocampal neuron cultures than the less pathogenic variant CVS-B2c. (Morimoto, K., et al., J. Virol. 73:510-517, 1999). The extent of apoptosis seen in neurons infected with the different viruses was associated with their levels of rabies virus G protein expression. CVS-B2c infection caused the expression of high levels of G protein and extensive apoptosis while CVS-N2c induced only minimal G protein production and limited apoptosis. The correlation of pathogenicity with cell death led to the speculatulation that the pathogenicity of a particular rabies virus may be dependent upon the capacity to avoid inducing a strong antiviral immune response. (Morimoto, K., et al., J. Virol. 73:510-517, 1999).
Unlike highly pathogenic rabies viruses, which fail to elicit a protective immune response, infection with weakly pathogenic tissue culture-adapted rabies viruses induces a strong antiviral response. In particular, rabies virus-specific cytotoxic T cells (Wiktor, T. J., et al., Proc. Natl. Acad. Sci. 74:334-338, 1977; Wiktor, T. J., et al., J. Ex. Med. 145:1617-1622, 1977) as well as G protein-specific VNA (Wandeler, A. I., et al., Rev Infect. Dis. 10 suppl. 4:649-653, 1988), which are considered to be the major effectors in the immune defense against a lethal rabies virus infection. (Cox, J. H., et al., Infect. Immun. 16:754-759, 1977). Therefore, virus-induced cell death may make an important contribution to the stimulation of the rabies virus-specific immune response. In this context, it has been suggested that virus-induced apoptosis may have a physiological role in protecting the CNS from progression of infection and allowing contact between virus and immune components. (Galelli, A., et al., J. Neuro Virol. 6:359-372, 2000). Thus, the enhanced immunogenicity of attenuated rabies virus strains could be associated with increased cell death.
While apoptosis occurring under certain physiological conditions, such as during development, is an immunologically innocuous event, apoptotic death after viral infection or ligation of Fas can trigger powerful innate and adaptive immune responses. (Restifo, N. P., Current Opinion in Immunol. 12:597-603, 2000). The possibility that cells undergoing apoptosis induce signals that enhance the immune response to the virus is supported by findings demonstrating that cell injury releases endogenous adjuvants that stimulate cytotoxic T cell responses. (Shi, Y., et al., Proc. Natl. Acad. Sci. 97:14590-14595, 2000). Furthermore, apoptotic cells can induce maturation of dendritic cells and stimulate their presentation of antigen to both class I- and class II-restricted T cells. (Chattergoon, M. A., et al., Nature Biotechnology 18:974-979, 2000; Rovere, P., et al., J. Immunol. 161:4467-4471, 1998). The present invention relates to the ability of less pathogenic rabies viruses to cause an increase in cell death, thereby inducing an immunogenic response against the rabies virus.
The invention disclosed herein relates to the construction of a recombinant rabies virus expression vector that expresses a pro-apoptotic protein, for example cytochrome c, thereby stimulating antiviral immunity against the rabies virus. (Schnell, M. J. et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; Schnell, M. J. et al., EMBO J. 13:4195-4203, 1994) cytochrome c is essential for the proteolytic activity of Apaf-1 and the activation of caspases (Harvey, N. L. & Kumar, S., Adv. Biochem. Engineering-Biotechnol. 62:107-128, 1998) and causes an acceleration of apoptotic cell death if overexpressed. (Bradham, C. A., et al., Mol. Cell. Biol. 18:6353-6364, 1998). The modular genome organization of the rabies virus readily allows genetic manipulations of viral genes and stable expression of large foreign genes up to 4 kb. (Schnell, M. J., et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000). Cytochrome c is used herein as an example of a pro-apoptotic protein, but it is obvious to those of skill in the art that variations in the pro-apoptotic protein may be used, and it is intended that the invention may be practiced otherwise then as specifically described herein. Examples of pro-apoptotic proteins that are also used in the present invention include, but are not limited to, Bad, caspase, Bok, Bax, Bak, Fas, etc. Cytochrome c plays a role in the induction of nuclear apoptosis and is highly conserved between species such that any effect on the immunogenicity of a rabies virus vaccine strain in mice will be applicable to other target species. The expression of cytochrome c by a rabies recombinant virus of the present invention will accelerate cell death, enhance immunogenicity, and attenuate pathogenicity of the rabies virus.